I. NEMO Syndrome NEMO, a non-catalytically active component of the IkappaB alpha kinase (IKK) complex, is required for IKK enzymatic activity and localization. In its absence nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) transcription factor family members do not translocate to the nucleus to regulate gene transcription, and normal development of the organism, including the immune system, does not occur. We apply a forward genetic approach as the basis of our analysis, in that specific mutations which result in NEMO immunodeficiency and inflammatory disease are investigated using biochemical, cell biological, and molecular techniques. In addition to clinical and laboratory evaluation of human patients these studies are conducted in patient derived induced pluripotent stem cells, and a reconstitution system utilizing NEMO-deficient Jurkat T cells. Because NEMO syndrome is associated with defects in development of ectodermal structures in addition to secondary lymphoid structures in association with certain mutations, we seek to understand the function of NEMO in both of these roles in addition to its role in hematopoietically derived cells. We have generated human iPSC to study the effects of mutation on the development and function of these various cell types. NEMO not only is required for IKK activation, but is also thought to serve as a scaffold to other signaling proteins. Its regulation is complex: splice isoforms exist, and it is post-translationally modified by serine phosphorylation, non-degradative ubiquitination, sumoylation. We recently found that particular C-terminal truncation mutations that increase NF-kB activation do not do so by increasing IKK kinase activity, suggesting impairment in a secondary regulatory function. In addition to being directly modified, NEMO recognizes different ubiquitin chains via its central UBAN motif and the C-terminal Zinc Finger (ZF). C terminal truncation has been shown in vitro to shift the affinity of the centrally located UBAN ubiquitin binding motif from K63 to linear ubiquitin. We have developed an expression system to express mutant forms of NEMO and determined quantitative binding affinities to linear (M1) and K63 linked polyubiqutin by different mutant NEMO that result in inflammatory disease. We hope to gain insight into this potential affinity switch mechanism which may determine how signaling via distinct receptors such as Toll-like receptors and TNFR can induce increased NF-kB transcription whereas TCR-induced transcription is blocked. Variability of clinical disease phenotypes and immune-function lab abnormalities make management of individuals with NEMO syndrome and inflammatory disease difficult. Because of our incomplete understanding, treatment paradigms, such as the use of allogeneic hematopoietic stem cell transplantation, cannot at this point be standardized and often result in poor outcomes. We are establishing protocols to evaluate patients at the Clinical Center to be able to comprehensively characterize the spectrum of phenotypes, and are working to develop protocols to offer advanced treatment based on our clinical and laboratory findings. In a similar fashion, we are studying individuals with NEMO-LIKE Syndrome, who have suspected monogenic diseases which phenocopy NEMO syndrome in some aspects. We hypothesis that these individuals will have defects in signaling proteins or other regulatory proteins that interact in the NF-kB signaling pathway. Discovering the defects that cause the disease will yield insight into immunity and inflammatory disease that will allow us to diagnose and treat more common disorders. II. WASp role in iTreg signaling Recent work indicates a role for the inhibitor of NF-kappaB kinase complex member, IKKbeta, in the subcellular localization of signaling proteins (HOMER-3) which mediate TCR signaling through mediating cystoskeletal changes and whose normal function is required to prevent autoimmunity. Recently, work has shown that iTreg development is abnormal in individuals with WAS mutations, and that c-Rel is required for normal development of this subset. Work is currently being done to evaluate the role of WASp deletion in mice on iTreg development and NF-kB function. In WAS deficient iTreg, we have found that CD3 stimulation results in 20-fold higher c-Rel cytoplasmic levels compared to normal control, but that this does not lead to increased c-Rel nuclear levels or binding following costimulation with CD28. We will continue to investigate how WASp deficiency leads to NF-kB signaling defects and how this influences T cell subset development and function.